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Calcium Phosphate Transfection of Neurons in Primary Culture

Calcium Phosphate Transfection of Neurons in Primary Culture

Developed by Hank Dudek and Zhengui Xia (11/96)
Modified by Steve Finkbeiner (rev. 7/97)
Departments of Neurology and Physiology, UCSF/GIND
415-695-3868; sfinkbeiner@gladstone.ucsf.edu

  1. Protocol
    1. make calcium phosphate/DNA precipitate:
      1. variables
        • volume of ppt
        • 24 well: 20-40ul
          60mm plate: 120-200ul
        • amount of DNA :
        • 24 well: 2-4ug (eg., for immunostaining)
          60mm: 2-5ug (eg., for RNAase protection)
      2. recipe: eg., for 300ul precipitate (scale up or down accordingly):
        1. in a 15ml polystyrene pop-cap tube, mix DNA and CaCl2 (1/2 final volume, therefore 150ul in this case)
        2. 15ul 2.5M CaCl2 (1/10 of DNA/CaCl2 volume)
          DNA (eg., 30ug = enough for 10 wells at 30ul ppt per well)
          sterile H2O to 150ul

        3. aliquot 2X Hepes-Buffered Saline (HeBS) to second tube (150ul, in this case)
        4. add DNA/CaCl2 to 2X HeBS dropwise with pipetman, while swirling 2X HeBS
        5. let ppt form in dark, 25', room temp
        • note: typical plan:
          # wells:10
          total vol ppt:300ul
          DNA/CaCl2 vol:150ul
          2.5 M CaCl2:15ul
          plasmid DNA:X ul (=30ug)
          sterile dH2O:X ul (to 150ul)[add to tube first]
          2X HeBS:150ul
        • [make extra volume]
    2. replace culture media with transfection media (37C) (near end of 25' ppt formation time)
    3. Add ppt to plates
    4. Stop transfection
      1. aspirate media, wash twice with fresh transfection media(37C)
        • volumes: 24 well: 500ul; 35mm: 1ml; 60mm: 2ml
      2. add back conditioned media
        • optional: containing 100uM APV [NMDA receptor antagonist]
        • 24 well: 300ul; 35mm: 1.5ml; 60mm: 2ml
        • from start of procedure, or other appropriate conditioned media
        • if necessary, bring conditioned media to sufficient volume with culture media
  2. Reagents/solutions
    1. transfection media: either (see notes):
      1. MEM
        • Gibco/BRL# 12370-037 with Hanks' salts
        • with 25mM Hepes
          without L-glutamine
        • bring to pH 7.85 with NaOH; sterile filter
        or
      2. DMEM
        • Gibco/BRL #11960-028
        • note: for (i) or (ii): no added serum, Penn-Strep, or glutamine
        • optional (see notes): include Kynurenate-Mg: 10 volumes transfection media plus 1 vol. 10X Ky-Mg
        • 10 X Ky/Mg stock (10 mM Kynurenic acid/100 mM MgCl2)
          dH2O170 ml
          Kynurenic acid378 mg
          0.5 % Phenol Red1 ml
          1 N NaOH1.8 ml
          1 M Hepes (Sigma)1 ml
          1 M MgCl220 ml
          • stir to dissolve, gradually adjusting pH to 7.4 (by eye) with 1 N NaOH
          • add dH2O to 200 ml total
          • Filter sterilize (in hood)
          • aliquot to 30 ml; store @ -20 C
          • as thaw aliquots, store at 4 C, up to 1 mo.
  3. 2X HeBs
  4. final conc200 mlsupplier
    NaCl274 mM3.2 gBaker # 3624-05; Mallinckrodt
    KCl10 mM142 mgMallinckrodt # 6858
    Na2HPO4.7H2O1.4 mM76 mgMallinckrodt # 7914; 268g/mol
    dextrose (D-glucose)15 mM540 mgBaker # 1916-01; 180g/mol
    Hepes (free acid)42 mM2 gCalbiochem # 391338; 238g/mol
  • Notes
    1. Amount of precipitate (ppt) per plate, length of time ppt on plates
    2. Transfection media
    3. The wash with transfection media (before placing the cells in transfection media) seems to be important for removing a residual media component that inhibits precipitate formation/accumulation. This component may be serum, and therefore the wash may be unnecessary if cells are grown in serum-free media.
    4. Channel inhibitors
    5. Amount of DNA
    6. Glycerol shock
    7. Cell culture
    8. amt of CaCl2 in pptn reaction
  • Ref.s :
    1. Xia, Dudek, Miranti, and Greenberg, J. Neurosci. 16, 5425-5436, 1996.
    2. Bonni, Ginty, Dudek, and Greenberg, Molec. Cell. Neuroscience, 6, 168-183, 1995.
    3. Nikolic, Dudek, Kwon, Ramos, and Tsai, Genes & Dev. 10, 816-825, 1996.
    4. Dudek, Datta, Franke, Birnbaum, Yao, Cooper, Segal, Kaplan, and Greenberg, Science,      in press.